Recombinant adeno-associated virus (rAAV) vectors efficiently deliver therapeutic genes in many target tissues. They have become very popular and are widely used for many gene transfer experiments including gene therapy of metabolic diseases and genetic disorders. Since rAAV vectors are generated based on a non-pathogenic virus, they have been considered as one of the safest vectors for years. However, recent reports of unpredictable retroviral insertional mutagenesis in mice and human subjects, and our recent findings in an analysis of a limited number of rAAV integration events showing that rAAV vectors preferentially integrate into active genes, have raised concerns about the potential for rAAV-mediated insertional mutagenesis. Therefore, it is very important to re-define the risk of rAAV-mediated gene therapy. Our long-term objectives in this regard are to establish the risk of rAAV vector integration and to develop the safest rAAV vector systems. To achieve these goals, we propose three exploratory studies. First, we will develop a novel high-throughput method for isolation of rAAV vector insertion sites in vector-transduced tissues and carry out a large-scale analysis for thorough understanding of rAAV vector biology and consequences of rAAV vector integration. Second, considering that rAAV preferentially integrate into active genes, we assume that continuously regenerating cells due to hepatocellular injury may have dysregulated expression of cell cycle-related genes, which may be susceptible to rAAV vector integration, leading to accelerated malignant transformation of hepatocytes. Therefore, we will assess synergistic procarcinogenic potential of rAAV vectors using transgenic mouse models for viral hepatitis predisposed to liver cancer. Third, we will explore the use of mutant Cre recombinases (retaining the ability of IoxP site binding but catalytically inactive) to develop a non-integrating rAAV vector system. We hypothesize that expression of a mutant Cre and the use of rAAV vectors with a IoxP site at each vector end, will facilitate intramolecular selfcircularization and ultimately inhibit rAAV vector genome integration. We will establish proof of principle of this approach with a series of in vitro and in vivo experiments. The results from these proposed studies will lead to profound understanding of the mechanisms of rAAV vector integration and the risk of insertional mutagenesis, and to development of new technologies to avoid unwanted vector genome integration.